1. Field of the Invention
The invention relates to a pharmaceutical composition capable of stimulating the hematopoietic function so as to mobilize and give rise in the circulation to extremely high levels of stem cells originating from the bone marrow. This invention makes it possible to offer:
1) new antimyelotoxic therapies; PA1 2) new methods for harvesting bone marrow stem cells, making it possible to replace marrow biopsies for the purpose of: PA1 1) to counteract the myelotoxic effects of certain drugs in order to maintain a normal level of blood cells (especially of neutrophils), PA1 2) to replace bone marrow biopsies by the harvesting of high levels of HSC in the circulating blood for the purpose of homologous or autologous grafting of marrow cells, PA1 3) to obtain from the circulating blood a high level of HSC in order to use them as vectors for carrying out certain types of gene therapy. PA1 Nac--Mur--L--Ala--D--Glu[OMe]--OMe.
a) autologous or homologous grafting, PA2 b) use in gene therapy techniques.
Said pharmaceutical composition comprises at least one MDP derivative of general formula (I) below, and an especially impressive example of which, for the subject of the invention, is Muradimetide of formula Nac--Mur--L--Ala--D--Glu--[OCH3]--OCH3 or MDPA dimethyl ester.
2. Description of the Related Art
Muramyl peptides and their ester derivatives are a family of molecules which have been described in numerous publications and numerous patents since the first description of N-Acetyl-Muramyl-L-Alanyl-D-isoGlutamine (MDP for muramyl dipeptide). Briefly, they are capable of exerting a wide variety of biological activities including an immunoadjuvant activity, an anti-infectious activity, an antitumor activity, an antiallergic activity and a regulation of cytokine secretion. However, a number of these muramyl peptides, while they can display most promising biological activities, often display side effects or drawbacks which prevent their use as a medicament, in particular toxic effects or a difficulty of formulation on account of their low solubility in aqueous solutions.
They display, in addition, a wide variability in their spectrum of action.
Very recently, it has been shown that the muramyl peptide diesters of general formula (I) below, and especially Muradimetide, possess the special property of being immunostimulatory, which has simultaneous advantages of efficacy, of formulation and of lack of toxicity, a plurality of advantages which had not been observed for other MDP derivatives; in particular, an immunomodulator which is active by external administration, in particular by oral administration by aerosol, can enable a wider and more flexible use of this immunostimulant to be envisaged; the advantage of good tolerability also occurs when this derivative is administered systemically.
The essential features of the properties already described for MDP derivatives are linked to a strengthening of the humoral or cellular immune system.
Surprisingly, the present invention is the outcome of the discovery according to which some types of MDP derivatives, represented below by the formulae (I), (II) and (III) in combination, where appropriate, with a compound of formula (IV) or the conjugates of formulae (V) and (VI), are capable of increasing the level of normal and immature formed elements of the blood, and especially of neutrophils in the circulating blood, in particular after administration of doses of AZT likely to decrease considerably the level of circulating leukocytes. In fact, after administration of the pharmaceutical composition, an increase in normal leukocytes is observed in the circulation, accompanied by a very large increase in myelocytes/promyelocytes, followed by an increase in the neutrophils and, later, in the lymphocytes. Most remarkably, this effect is observed very strongly even after a combined treatment involving highly myelotoxic doses of AZT.
All blood cells are derived from common stem cells whose origin is the bone marrow. Before reaching their final stage of differentiation, these cells pass through different steps: pluripotent stem cells can differentiate either into T or B prolymphocytes, or into stem cells of the erythrocytic, myelocytic, granulocytic and myelokaryocytic lines. In these different intermediate steps, they are not morphologically discernible and are designated by the terms promyelocyte/myelocyte for the youngest and metamyelocytes for those closest to differentiation.
In the text hereinafter, these undifferentiated cells will be designated collectively HSC for "hematopoietic stem cells".
In the normal state, the level of HSC is of the order of 1% of the white cells present in the blood.
It is advantageous to activate hematopoiesis and to mobilize HSC in the following three cases:
1) Antimyelotoxic treatment.
Some treatments or medicaments give rise to myelotoxic effects, such as chemotherapies for cancer or for AIDS, immunotherapies or radiotherapies, leading to a dysfunction of the hematopoietic function and thereby dramatically increasing the risks of intercurrent infections. The pharmaceutical composition of the invention enables the decrease in the cells of the immune system resulting from this myelotoxicity, and especially in the neutrophils, to be counteracted.
In addition, some treatments which employ immunotherapy, in particular by cytokines, can also give rise to a cytotoxic effect for which the compositions of the invention can play the part of an antagonist.
2) Obtaining of cells enabling bone marrow grafting to be performed.
Anticancer treatments by severe chemotherapy or by radiotherapy give rise to a destruction of the cells of the blood and the cells of the immune system. In these cases, the hematopoietic system is reconstituted by grafting bone marrow cells. Autologous grafts are performed when the cancer affects cells other than those of the marrow and, in this case, samples are taken from the patient himself before the destructive treatment. Homologous grafting (that is to say using cells originating from a compatible donor) is performed when the marrow cells are themselves cancerous.
In both cases, it is extremely advantageous to be able to enrich the bone marrow with stem cells, but most particularly to be able to obtain the latter in the circulating blood in a sufficiently high number to facilitate their use as grafts. In fact, at the present time, the use of blood for the preparation of grafts enriched with HSC requires the taking of a large number of large samples, which is hence spread out over a long period; furthermore, it is necessary to implement a preparation which is distressing for the patient and for the donor, since enrichment with HSC is carried out by administration of expensive and sometimes poorly tolerated cytokines (GM-CSF), followed by the taking of a large number of large blood samples, the collective treatments necessitating hospitalization every time. The pharmaceutical composition of the invention enables the material needed for marrow grafting to be obtained in ambulatory subjects and after only a few days of treatment, since it is capable of inducing in the blood, after approximately 4 days of treatment, a multiplication of the number of HSC by 200.
3) Obtaining of readily purifiable stem cells which can be used for gene therapy.
The use of certain forms of gene therapy techniques will depend on the possibility of obtaining HSC in large amounts in the circulating blood. This objective can be achieved by means of the pharmaceutical composition of the invention, enabling an extremely large percentage of HSC to be obtained in the blood, whereas these cells are very difficult to purify from the bone marrow.
Preclinical experiments and clinical trials of gene therapy showed that it was necessary to use stem cells which have a long life and a high power of expression and which distribute themselves throughout the body. It is very generally accepted that HSC are especially advantageous gene vectors, either for counteracting immune deficiencies such as Gaucher's disease or cases of profound immune deficiencies (SCID for severe combined immune deficiencies), or for counteracting the development of cancers such as myeloma, leukemia, cancer of the breast or ovary, and the like, or for inducing a resistance to infectious diseases, especially AIDS.
To meet these objectives, it is hence essential to have at one's disposal a large number of HSC which can be readily purified from the circulating blood by the usual cell separation techniques, and this is precisely the situation afforded by the composition of the invention or the medicaments containing it.
The composition of the invention makes it possible to envisage the development of the use of the cells of the circulating blood to reconstitute the hematopoietic function after cellular destruction, as a result of a multiplication, which can reach a factor of 200, of the percentage of circulating HSC, whereas, in the absence of treatment, not more than 1% of these cells can be detected.
This effect on the HSC occurs irrespective of the stage of differentiation of said stem cells, as shown in Example 7 below.